Electroretinogram (ERG) to Evaluate the Retina in Cases of Retinitis Pigmentosa (RP)

Abstract

Electroretinogram (ERG) captures the electrical responses of photoreceptors, the summation of action potentials from all neurons in the retina elicited by illumination. ERG testing is an incredibly useful tool in obtaining more specific information regarding a retinal dystrophy. Specifically, ERGs are typically used to test photoreceptors and inner retinal function in humans and animals, to diagnose retinal dystrophies, and to monitor disease progression. In this chapter, we will introduce the components of ERGs and the standard ERG protocols for clinical examination. We will also introduce the various specialized ERG tests, which can help to differentiate retinitis pigmentosa (RP) from other retinal disorders. Lastly, we will elaborate on how to use ERGs to predict visual prognosis in RP.

1. Introduction

1.1. Overview of the Visual System

The visual system is comprised of the retina, visual pathways from the retina via the lateral geniculate nucleus (LGN) to the primary visual cortex, and other brain areas involved in visual information processing. First, light signals are converted into electrical signals in the photoreceptors of the retina. These signals are sent to interneurons, such as bipolar cells, horizontal cells, and amacrine cells, and then to ganglion cells. Transmitted down the axons of the ganglion cells through the optic nerves, the signals travel down the optic tracts, across the optic chiasm, until they reach the LGN. The primary visual cortex receives signals from the LGN, and those signals are further processed in the visual associative cortex.

1.2. Historical Perspective

Dewar (1877) was the first to successfully perform an ERG on humans [1]. However, using Dewar’s design, it was impossible to derive quantitative measurements because eye movements resulted in baseline disturbances. Therefore, these difficulties limited the demand for this test in the clinical setting. Kahn and Löwenstein (1924) recorded the ERG by placing a string galvanometer and leads over an anesthetized eyeball; however, this was fraught with difficulties [2]. Granit (1934) initially examined the ERG waveforms in detail and correlated each component of the ERG (P-I, P-II, and P-III) to individual cells in the retina. Today, a-wave (the large negative component PIII), b-wave (the development of positive component PII followed by the resulting PIII and PII), and c-wave (the summation of P-I and P-III) have long been used in ERG interpretation, which are still based on Granit’s analysis. Granit was awarded the Nobel Prize in Physiology or Medicine in 1967 for his discoveries of the primary physiological and chemical visual processes in the eye [3]. With advances in technology, Lorrin Riggs (1941) introduced the design of the contact lens electrode, which made it possible to employ the ERG as part of a routine examination in a clinical setting [4]. Moreover, a better understanding of the components of the ERG facilitated its use in clinical applications.

1.3. The Components of ERG

ERG responses represent a mass response generated from all types of cells in the retina. In other words, all retinal cell types can contribute to the responses of ERGs. While some cells make a large contribution to the ERG waveforms, others have a negligible effect. Different orientations of these cells might result in different responses to a change in illumination [5]. For example, radial voltage current flow in extracellular media from radially oriented cells will sum up, while lateral currents will be offset by each other because of the incredible symmetry in retinal lateral arrangement [6]. Photoreceptors are the first in the retina to be activated by light and together with bipolar cells are major contributors to major waves in ERG responses. Therefore, an initial negative a-wave is mainly generated by photoreceptors and followed by a positive b-wave, produced from the OFF- and ON-bipolar cells, respectively. Compared to photoreceptors and bipolar cells, horizontal and amacrine cells make fewer contributions to the waveforms captured by ERG as a result of their lateral orientation [5].

1.3.1. The Origin of a-Wave

The a-wave is mainly generated by photoreceptors, but it also relates to the post-receptoral pathway. The dark-adapted a-wave, an initial cornea-negative deflection that occurs in response to light stimuli after dark adaptation, is primarily driven by rods (with or without cones depends on the light intensities). The light-adapted a-wave, an initial negative wave in response to a flash of light after light adaptation, is generated mainly by the cone photoreceptors since the activity of rod photoreceptors is inhibited [7].

1.3.2. The Origin of b-Wave

After the first negative deflection, the ensuing positive deflection is b-wave. The major generators are ON-bipolar cells and Muller cells. On the one hand, the b-wave is generated from the voltage drop of extracellular current resulting from radial current flow through the ON-bipolar cells. On the other hand, a release of potassium produced by the depolarizing ON-bipolar cells causes depolarization of Muller cells, resulting in a cornea-positive deflection [8–12]. In clinical application, the b-wave is an indicator to assess inner retinal function or signal from photoreceptors.

1.3.3. The Origin of c-Wave

In the subretinal space, a reduction in the concentration of potassium ion results in the generation of the c-wave, the process that leads to apical polarization of the retinal pigment epithelium (RPE) [3]. Also, normal c-wave relies on the unimpaired photoreceptors, which trigger a reaction chain to decrease in the extracellular concentration of potassium ions when they respond to light. The ERG c-wave can be used as a tool to assess the RPE function, the integrity of the photoreceptors, and the interactions between them [6].

1.3.4. The Origin of d-Wave

In the retina, not only the onset of light but also the cessation of light elicits the responses, which are called ON-response and OFF-response, respectively. The d-wave is a positive-going deflection in the photopic ERG, derived from strong hyperpolarizing (OFF) cone bipolar cells at light offset [7].

2. Materials

2.1. Types of Recording Electrodes

2.1.1. Recording Electrodes

Active ERG electrodes are connected to the positive input for ERG recording, and all of them can be placed on the cornea, bulbar conjunctiva, or skin of the lower eyelid. These include Burian–Allen electrode (contact lens electrode, bipolar electrode), Henkes Lovac (contact lens electrode), Goldlens (contact lens electrode), ERG Jet (contact lens electrode), gold foil electrode (lid-hook electrode), C-glide (lid-hook electrode), HK loop (lid-hook electrode), Dason–Trick–Litzkow (DTL) electrode, and skin electrodes [13].

Contact lens electrodes give more reliable and reproducible recordings and provide the highest amplitudes compared to other skin or lid electrodes. The Burian–Allen electrode is most commonly used because it holds the lid apart to prevent blinking and lid closure [14], and it has different sizes for premature infants, children, and adults. However, it also has some disadvantages, such as low tolerance in some people due to irritation and risk of corneal or conjunctival abrasions. In such cases, it is usually necessary to use topical anesthetic eye drops and 0.5 or 1% methylcellulose solution over the corneal surface to protect cornea before the insertion of Burian–Allen electrode [13, 15, 16].

The Dawson–Trick–Litzkow (DTL) electrode is the second most popular electrode after the Burian–Allen type. The design of the DTL electrode is based on a nylon thread whose individual fiber is silver-impregnated, and the conductive thread is placed in the inferior fornix for ERG recording. Compared to contact lens electrodes, the DTL electrode provides better patient tolerance. If the wrong placement of the thread occurs, the recording might fail because of induced eye movements, such as blinking and eyelid closure. In general, the amplitudes recorded using the DTL electrode are about 10% lower compared to the amplitudes recorded using contact lens electrodes [15, 16].

Skin electrodes are usually not recommended as standard recording electrodes for adults but may be considered in infants and young children who cannot tolerate irritation from contact lens electrodes. The signals recorded using skin electrodes are usually smaller, more variable, and much noisier than signals recorded using corneal contacting electrodes [16].

2.1.2. Reference Electrodes

Reference electrodes are connected to the negative input of the system. In bipolar electrodes, the lid speculum covered by conductive material over its outer surface serves as a built-in reference electrode. The contact lens–speculum assembly provides a more stable signal in recording than monopolar contact lens electrodes with a separate reference [17]. Alternatively, skin electrodes are placed temporally near the orbital rim or on the forehead. Moreover, it should be noted that electrical activity produced by skeletal muscles might interfere with ERG recordings if the reference electrodes are placed over muscle masses.

2.1.3. Common Electrode

The common electrode type is connected to the common input of the recording system. Typical locations for common electrodes are the earlobe, mastoid, and forehead.

3. Methods

3.1. The Clinical ERG Procedures

The human ERG recorded at the cornea and elicited by a full-field stimulus is a mass response generated by cells across the entire retina. To get reproducible amplitudes and implicit times in the ERGs, the flash stimuli and background light should be homogeneous, and all reach over around the entire retina. Therefore, all of the receptors are stimulated or adapted in the same condition.

Clinical electrophysiology tests include ffERG, electrooculogram (EOG), multifocal ERG (mfERG), pattern ERG (pERG), and visual evoked potential (VEP). Each test is intended to test specific cell function in the visual system and provides information to support clinical diagnosis or function to predict prognosis. Correlation between each test and function is summarized in Table 1.

Table 1.

Clinical use of the various electrophysiological tests

Type of test	Clinical use of the various electrophysiological tests
ERG	Assess the functions of photoreceptors and neurons in the inner nuclear layer
EOG	Assess generalized retinal pigment epithelium (RPE) function
mfERG	Reflect macular and central cone function
Pattern ERG	Reflect retinal ganglion cell function in macula
VEP	Quantify nerve function from retina to visual cortex, such as optic nerve, optic chiasm, and retrochiasmal pathways

The International Society for Clinical Electrophysiology of Vision (ISCEV) proposed several standard settings for these tests [15, 18, 19], which allow physicians and scientists to communicate and discuss the visual electrophysiological function using the same settings.

3.2. The ISCEV Standard for Clinical ERG

The ISCEV Standard is reviewed regularly. In this chapter, we used the most recently published versions of the ISCEV Standard protocols (2015, 2018). The ISCEV Standard ERG includes six steps for ffERG, which are named after the adaptation state and the flash intensities (stimulus calibrations in cd s/m²) [20].

1. Dark-adapted 0.01 ERG (rod ERG).

2. Dark-adapted 3 ERG (combined rod-cone standard flash ERG).

3. Dark-adapted oscillatory potentials.

4. Dark-adapted 10 ERG (strong flash ERG).

5. Light-adapted 3 ERG (standard flash “cone” ERG).

6. Light-adapted 30 Hz flicker ERG.

3.3. The Standard Procedure for Performing ERGs on Patients

1. Pupillary dilatation. To get fully dilated pupils, both tropicamide (relaxes the circular muscles of the iris) and phenylephrine (contracts radial muscles of the iris) are used for mydriasis. According to the ISCEV Standard, the sizes of the pupils should be measured before and after the ERG recordings.

2. Dark adaptation. Dark adaptation for at least 20 minutes is necessary before this first step in ERG examination.

3. Insertion of corneal contact electrodes after dark adaptation. Electrodes are inserted under dim red light rather than strong red light, and then an additional 5 minutes recovery time is recommended. (see Note 1).

4. Fixation for ERG recording. Patients should be instructed to look at a target point and avoid eye movements. Eye movements might change the positions of the electrodes on the eye, and blinking or closing the eyelids might cause large artifacts.

5. Dark-adapted 0.01 ERG (rod ERG). This is the first step after dark adaption, and the weak flashes should be tested before the strong flashes. The a-wave is not detected because the signal from the rod photoreceptors is too small under the dim stimuli, but the b-wave is elicited when the signals are amplified by around 100-fold through the rod-initiated pathways [15, 21].

6. Dark-adapted 3 ERG (combined rod-cone standard flash ERG). The dark-adapted 3 ERG is the second step after the dark-adapted 0.01 ERG. Under these stimuli (3 cd·s/m²), the a-wave and b-wave can be recorded by activation of photoreceptors and by ON- and OFF-bipolar cells, respectively.

7. Dark-adapted oscillatory potentials. After dark adaptation, the dark-adapted oscillatory potentials (OP) are recorded with the flash strength of 3.0 cd·s/m². The extraction of the OPs might be from an intact ERG. Amacrine cells (or inner retina, such as bipolar cells) are involved in the generation of the OPs [15, 22].

8. Dark-adapted 10 ERG (strong flash ERG). Compared to the dark-adapted 3 ERG, the dark-adapted 10 ERG features the increase in the amplitudes of a-wave and b-wave, the shortening peaking time (greater distinction of negative ERG waveforms, when b-wave amplitudes are reduced), and the emerging of oscillatory potentials at the ascending phase of the b-wave. These brighter flashes might give more reliable responses in patients with opaque media or immature retinae.

9. Light-adapted 3 ERG (standard flash “cone” ERG). Light adaptation for at least 10 minutes is required to maximize cone response and minimize rod activity. The a-wave in the light-adapted 3 ERG arises from the cone photoreceptors and OFF-bipolar cells and the b-wave from cone ON- and OFF-bipolar cells.

10. Light-adapted 30-Hz flicker ERG. The light-adapted 30-Hz flicker ERG is recorded with a flash strength of 3 cd·s/m². The 30-Hz flash stimulus reflects cone systems with post-receptoral ON- and OFF-pathways [15, 18].

See ISCEV Standard ERGs for details of stimulus strength, inter-stimulus time (rate), and recording bandpass (Hz) in the six steps.

3.4. ERG in Infants

It is challenging to record ERG responses from infants or young children because of the short attention span and poor cooperation. To alleviate their stress in the process, they need a quiet and calm environment accompanied by their primary caregiver [16]. Sometimes, it would be helpful to use sedation agents for noncompliant children, but the level of sedation might affect ERG responses.

3.5. Interpretation of ERG Responses

The amplitude and implicit time (or called peak time) of the a-wave and b-wave, as well as the latency of response, are important information for ERG analysis. The amplitude of the a-wave is a measure of the displacement from the baseline to the a-wave negative trough and that of the b-wave is from the a-wave negative trough to the b-wave positive peak. The implicit time is the time interval from the onset of the stimulus to its peak. The recording duration for single flash in a protocol is in the range of 250 millisecond (mS) [16] (see Fig. 1 for normal ERG responses).

Fig. 1.

Normal full-field electroretinogram (ffERG) responses. ffERG is recorded according to ISCEV standards. After a dark adaptation for at least 20 minutes, scotopic ffERG responses are elicited with different flash strength for dark-adapted 0.01 ffERG (a), dark-adapted 3 ffERG (b), dark-adapted 3 oscillatory potentials (c), and dark-adapted 10 ffERG (d). Following light adaptation for at least 10 minutes, photopic ffERG responses are elicited with certain flash strength for light-adapted 3 ffERG (e) and 30-Hz flicker stimuli for light-adapted 3.0 flicker ffERG (f)

To evaluate ERG responses, we should check for a change in amplitude (decreased) and implicit time (delay). The amplitudes of a-wave and b-wave correspond to the numbers of functional photoreceptors. The extent of retinal damage correlates to the degree of amplitude decrease. If some photoreceptors are damaged from focal lesions, such as retinal detachment, this might result in amplitude decrease without significant implicit time delayed in ERG responses. If the functions of photoreceptors are impaired in general, such as in rod-cone or cone-rod dystrophy, the amplitudes are usually decreased together with significant implicit time delayed.

Clinically, the ERG results using different equipment or from different labs/institutes might not be the same, even in a normal subject. Therefore, institutes/centers are suggested to establish their normative data for further ERG analysis. Clinicians can interpret the ERG results based on the electrode-specific and age-matched normative data. Also, test-retest reproducibility is important if clinicians plan to use ERG to monitor disease progression or test safety and efficacy after treatments [15, 16, 18].

3.6. The Typical ERG Findings in Patients with RP

In RP patients, their symptoms and clinical findings vary, even in those patients carrying the same gene variants or from the same family. RP falls into the category of “rod-cone dystrophy,” which means the rod function is affected more than cone function. Generally, RP is characterized by rod photoreceptor dysfunction in the early stage and progressive rod and cone dysfunction in more advanced stages [16]. Typically, the dark-adapted 0.01 ERG (rod responses) shows the reduced amplitude and delayed implicit time of b-wave. The dark-adapted 3.0 ERG and dark-adapted 10.0 ERGs (combined rod-cone response) show reduced a-wave amplitude, which indicates impaired rod photoreceptor function. The b-wave amplitude is reduced because it is derived from ON-bipolar cells, which are connected to the abnormal rod photoreceptors. The light-adapted 30-Hz and light-adapted 3.0 ERGs (cone response) are typically delayed and/or reduced, but cone function is less affected than rod function (Fig. 2). With progression of the disease, the responses could be small or even nondetectable in severe or end-stage RP patients [16].

Fig. 2.

Abnormal full-field electroretinogram (ffERG) responses for retinitis pigmentosa (RP). A patient in the early stages of rod-cone degeneration is illustrated by a decreased b-wave amplitudes in scotopic and photopic ffERGs (arrow) as well as delayed implicit times of the b-wave. In moderate to severe stages of degeneration, the responses are small or extinguished

3.7. The Specialized Protocols for Differential Diagnosis

Although ffERG is used to evaluate the whole retinal function, ISCEV encourages more extensive protocols for a specific clinical condition. Clinicians should be familiar with these protocols and select suitable protocols based on patients’ clinical presentation. In clinical practice, some diseases present with similar symptoms and signs as RP; however, the information from ffERG is not sufficient to make the correct diagnosis, which will require specialized protocols. In this section, some specialized ERG protocols will be introduced.

3.8. Prolonged Dark Adaption

Patients with some hereditary retinal dystrophies, such as fundus albipunctatus and retinitis punctata albescens, present with retinal white dots and night blindness. Using ISCEV Standard ERG, we cannot distinguish between these two diseases. Fundus albipunctatus is characterized by multiple yellowish white dots with sparing of the macula. Compared to RP, fundus albipunctatus is stationary or relatively slowly progressive. A prolonged 2.5-hour dark adaptation can improve rod response in fundus albipunctatus (Fig. 3) [18, 23], while there is still abnormal (no change) in retinitis punctata albescens.

Fig. 3.

Full-field electroretinogram (ffERG) recovery in fundus albipunctatus (FA) after prolonged dark adaptation. This FA patient underwent ffERG testing, revealing responses that are representative to those of a typical RP patient in the left eye, following impaired rod function in the left eye. The rod response of right eye recovered after prolonged dark adaptation (2.5 hours)

3.9. S-Cone ERG

Patients with enhanced S-cone syndrome (ESCS) present with night blindness and retinal pigmentation, which can resemble RP. However, there are three unique features of ESCS in full-field ERG, which could be helpful to differentiate ESCS from RP. First, extinguished rod responses in dark-adapted 0.01 ERG. Second, the waveforms in dark-adapted 3 ERG are similar to those in light-adapted 3 ERG except the amplitudes, which indicate a cone cell dominant retina. Third, the amplitude of a-wave in light-adapted 3 ERG is larger than the amplitude in light-adapted 30-Hz flicker ERG [24]. Although genetic testing of the NR2E7 gene remains the gold standard for diagnosis of ESCS, clinicians could further test patients with the S-cone ERG, which show “enhanced” S-cone response compared to the normal population, thereby making a diagnosis before screening NR2E3 gene [24]. S-cone ERG is an additional ERG protocol to assess short-wavelength cone response. ISCEV provides an extended protocol for the S-cone ERG by Perlman et al. [25]. For more information on this protocol, refer to https://iscev.wildapricot.org/standards/.

3.10. Predict Visual Prognosis in Patients with Retinitis Pigmentosa

ERG can be used to evaluate the photoreceptor function in patients with RP and can identify early stages of RP by detecting a delay in implicit times of b-wave [20, 26]. Moreover, ERG can be used to predict visual prognosis. Berson (2007) proposed to use a 30-Hz cone ERG amplitude to predict the remaining visual function in RP patients. His technique includes using computer averaged consecutive narrow bandpassed (electronically filtered) cone responses to improve the signal-to-noise ratio, which allows to record down to 0.05 μV. A cone ERG actuarial table, based on 6553 visits of 1039 patients, showed an estimated number of years for RP patients with their 30-Hz cone ERG amplitude to decline to 0.05 μV (i.e., virtual blindness) [27].

ERG is useful in predicting visual prognosis when patients are anxious about how RP would develop or what should be expected for their future. However, there are some limitations. For example, different ERG recording settings, such as stimulus strength range, inter-stimulus time, or recording bandpass, might result in the variability of ERG responses in RP. Caution is needed when evaluating the remaining visual function and prognosis from ERG responses of RP patients.

4. Notes

1. It should be noted that proper placement of the electrodes is essential to get accurate and reliable ERG recordings and proper electrode cleaning is also required to prevent transmission of infectious agents [15].

2. Although the ISCEV recommends a minimum of 30-minute recovery time after fundus photography, optical coherence tomography, fluorescein angiography, and retinal imaging for ERG testing, it would be better to avoid using strong light for retinal imaging before ERG testing.

3. Many factors might account for the variabilities in ERG responses, such as age, gender, refractive error, pupil size, clarity of cornea or lens, depth of anesthesia, body temperature, duration of stimulus, time of dark adaptation or light adaptation, inter-stimulus time, systemic circulation, medication, and maturation of retina. All of these procedures should be standardized to minimize the artifacts [28].